title: The heteromultimeric debranching enzyme involved in starch synthesis in Arabidopsis requires both isoamylase1 and isoamylase2 subunits for complex stability and activity.
abstract: Isoamylase-type debranching enzymes (ISAs) play an important role in determining starch structure. Amylopectin - a branched polymer of glucose - is the major component of starch granules and its architecture underlies the semi-crystalline nature of starch. Mutants of several species lacking the ISA1-subclass of isoamylase are impaired in amylopectin synthesis. Consequently, starch levels are decreased and an aberrant soluble glucan (phytoglycogen) with altered branch lengths and branching pattern accumulates. Here we use TAP (tandem affinity purification) tagging to provide direct evidence in Arabidopsis that ISA1 interacts with its homolog ISA2. No evidence for interaction with other starch biosynthetic enzymes was found. Analysis of the single mutants shows that each protein is destabilised in the absence of the other. Co-expression of both ISA1 and ISA2 Escherichia coli allowed the formation of the active recombinant enzyme and we show using site-directed mutagenesis that ISA1 is the catalytic subunit. The presence of the active isoamylase alters glycogen biosynthesis in E. coli, resulting in colonies that stain more starch-like with iodine. However, analysis of the glucans reveals that rather than producing an amylopectin like substance, cells expressing the active isoamylase still accumulate small amounts of glycogen together with a population of linear oligosaccharides that stain strongly with iodine. We conclude that for isoamylase to promote amylopectin synthesis it needs to act on a specific precursor (pre-amylopectin) generated by the combined actions of plant starch synthase and branching enzyme isoforms and when presented with an unsuitable substrate (i.e. E. coli glycogen) it simply degrades it.
authors.affiliation: Department of Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Maria
authors.initials: M
authors.lastname: Sundberg
authors.forename: Barbara
authors.initials: B
authors.lastname: Pfister
authors.forename: Daniel
authors.initials: D
authors.lastname: Fulton
authors.forename: Sylvain
authors.initials: S
authors.lastname: Bischof
authors.forename: Thierry
authors.initials: T
authors.lastname: Delatte
authors.forename: Simona
authors.initials: S
authors.lastname: Eicke
authors.forename: Michaela
authors.initials: M
authors.lastname: Stettler
authors.forename: Steven M
authors.initials: SM
authors.lastname: Smith
authors.forename: Sebastian
authors.initials: S
authors.lastname: Streb
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Zeeman
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005816:Genetic Complementation Test; D006003:Glycogen / Q000096:biosynthesis / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D007517:Isoamylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D055550:Protein Stability; D021122:Protein Subunits / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013213:Starch / Q000096:biosynthesis; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D006004:Glycogen Debranching Enzyme System; D046912:Multiprotein Complexes; D021122:Protein Subunits; D013213:Starch; D006003:Glycogen; D007517:Isoamylase